============ CHAPTER 10.3 ===============

THE INFLUENCE OF TACROLIMUS ON ExPERIMENTAL

AUTOIMMUNE DISEASE Angus W. Thomson, Noriko Murase,

Michael A. Nalesnikand Thomas E. Starzl

INTRODUCTION

Four classes of immunosuppressive drugs have been used to treat human autoimmune diseases: corticosteroids, alkylating agents (cyclophosphamide

and chlorambucil), antimetabolites (azathioprine and methotrexate) and more recently, cydosporine A (CsA). Before the advent of CsA, which was approved by the US Food and Drug Administration (FDA) for the therapy of organ allograft rejection in 1983, the successful use of immunosuppressive drugs in autoimmune disease was restricted to a handful of disorders. These included systemic lupus erythematosus (SLE), rheumatoid arthritis, myasthenia gravis and nephrotic syndrome. The use of CsA, which selectively inhibits T -lym­phocyte activation and cytokine production has resulted in a higher rate of improvement than previously observed in those autoimmune diseases with a presumed T cell pathogenesis. These diverse disorders include psoriasis, atopic dermatitis, uveitis, insulin-dependent (type I) diabetes mellitus. primary bil­iary cirrhosis, aplastic anemia and lichen planus. In contrast. autoimmune dis­eases that are believed to be mediated primarily by autoantibodies. such as the autoimmune cytopenias. myasthenia gravis. SLE, Graves' disease and the glomerulonephritides are relatively resistant to CsA. In addition to the im­proved rate of response seen with CsA, opportunistic infections (a potentially serious complication of cytotoxic drug administration) are very rare in CsA-treated autoimmune disease patients.

Despite the successes achieved with CsA. several important problems still exist with respect to the immunosuppressive therapy of autoimmune diseases. These include: (1) an insufficient response rate; (2) disease recurrence follow­ing drug withdrawal (failure to induce tolerance); and (3) the associated risks of drug toxicity or excessive immunosuppression (including infectious com­plications and malignancy). With respect to CsA. the most common side ef­fects are nephrotoxicity (including arterial hypertension), cosmetic deformity with hirsutism and coarsening of the facies. hypercholesterolemia and an in­creased disposition to diabetes mellitus. Resolution of these difficulties has become the incentive for discovery of new immunosuppressive agents that act

Principles 0; Drug Development In TransplantatIOn and AutoimmUnity, edited by Ronald Lieberman and Asoke Mukherjee. © 1996 R.C. Landes Company.

172 Principles of Drug Development in Transplantation and Autoimmunity

specifically on components of the immune system that are involved in disease pathogenesis and not on "thera­peutically irrelevant" cells. A wide range of new can­didate drugs that act at different stages of lympho­cyte activation/ proliferation are currently under development (Table 10.3.1). Of these new agents, tacrolimus (formerly known as FKS06), that has a similar mode of action to CsA but is considerably more potent, has recently been approved (April 1994) by the FDA for the therapy of liver allograft rejec­tion. Its efficacy in the prevention of allograft rejec­tion is well documented; more germane to the au­toimmune disease field however, is the ability of tacrolimus to reverse established rejection-an immu­nopathologic process that shares cellular and molecular mechanisms with many autoimmune disorders. Pre­clinical evaluation of the efficacy and safety of tacrolimus in experimental autoimmune disorders is an important process in the evaluation of its thera­peutic potential. This chapter briefly reviews the mode of action of tacrolimus and its influence on experi­mental autoimmunity.

MODE OF ACTION OF TACROLIMUS

The powerful anti-lymphocytic and immunosup­pressive properties of tacrolimus (C44H69N012eH20; mw 822), a fungally-derived macrocyclic lactone, were first documented in 1987.1.2 Although totally distinct in structure from the fungal product and cyclic pep­tide CsA, tacrolimus shares many of the properties of the latter immunosuppressant. Both agents exhibit very similar molecular actions which result in the selective inhibition of C04> T helper (TH) lympho­cyte activation, cytokine-gene expression and conse­quently, lymphocyte proliferation. Like CsA, tacrolimus inhibits T cell activation mediated via the T cell re­ceptor (TCR)-C03 complex and the cell surface molecule C02. It is very effective in suppressing lym­phocyte proliferation in vitro at concentrations 100-fold lower than effective concentrations of CSA.2.4 T acrolimus inhibits the generation of cytotoxic T cells

in human mixed lymphocyte reactions but does not affect antigen recognition by cytotoxic T cells or the mechanism by which target cells are destroyed. 5

Tacrolimus does not appear to inhibit antigen pro­cessing or presentation by human monocytes at drug concentrations which strongly suppress T cell prolif­eration.6 Tacrolimus does not affect Cal. mobiliza­tion, phosphatidylinositol turnover, or protein kinase C (PKC) activities. It does, however, strongly and specifically inhibit expression of early T cell activa­tion genes encoding interleukin-2 (IL-2) (the main growth factor for T cells) IL-3, IL-4, IFN-y, granu­locyte macrophage-colony stimulating factor (GM-CSF) and c-myc:' On the other hand, recent studies have shown that tacrolimus may spare IL-10 (cytokine syn­thesis inhibitory factor) gene transcription by cloned murine T helper-2 (TH 2) cells in viero, whilst sup­pressing concomitant IL-4 mRNA production by these cellsJ Thus, diffirential interference with T cell cytokine gene expression may be an important mecha­nism whereby tacrolimus inhibits lymphocyte activa­tion and immune suppression is maintained.

Clues to the molecular actions of tacrolimus and CsA came from studies of their respective intracellu­lar binding proteins-FKS06 binding protein (FKBP) and cyclophilin, each of which is a cis-trans peptidyl prolyl isomerase.S• lO Although binding of the drug by its receptor (or "immunophilin") inhibits isomerase activity, the immunosuppressive effects of tacrolimus and CsA result from the formation of complexes be­tween the drug and its respective isomerase. I I Both the complexes of tacrolimus (FKS06)-FKBP and CsA­cyclophilin bind specifically to three polypeptides­calmodulin and the two subunits of calcineurin (a Ca2>-calmodulin activated, serine-threonine protein phosphatase). In each case, the interaction of the immunophilin appears to be with calcineurin. The drug-immunophilin complexes but neither drug nor either immunophilin alone, have been shown to block the Cal.-activated phosphatase activity of calcineurin. JO

Calcineurin thus appears to be the molecular target of the drug-immunophilin complexes.

Table 10.3.7. New immunosuppressive drugs with potential for therapy of autoimmune disorders

Agent

Deoxysperguahn FK506 (Tacrolimus) Rapamvcin Letlunomlde Mizonbine Mycophenolate moietil Brequlnar sodium SK&F 105685

Structure

semisynthetic polyamine carboxycyclic lactone carboxycyclic lactone isoxazole derivative imidazole nucleoside mycophenolic aCid derivative carboxvllc acid derivative azasplrane analog

level of action

macrophage iunctlon, cytotoxic T cells inhibits IL·2 production inhibits IL-2 action ? B cell suppression inhibits DNA syntheSIS inhibits DNA synthesis inhibits DNA synthesis ? induction oi suppressor cells

The Influence of Tacrolimus on Experimental Autoimmune Disease

The second, important observation was that the drug-immunophilin complexes block Ca2'-dependent translocation of the pre-existing, cytoplasmic com­ponent of the nuclear factor of activated T cells (NF-AT) to the nucleus. 12 The nuclear component of NF-A T is transcriptionally inactive in all cells other than activated T -lymphocytes, and is induced by signals from the TCR. Its appearance is not blocked by tacrolimus or CsA. It is now believed that tacrolimus and CsA block dephosphorylation of the cytoplasmic component of NF-AT which is required for its trans­location to the nucleus. In the absence of both nuclear and cytoplasmic components, binding of NF-AT to DNA and transcriptional activation of the IL-2 gene is suppressed.

EFFECTS OF TACROLIMUS ON IMMUNE REACTMTY

The very potent inhibitory effects of tacrolimus on humoral and cell-mediated immune responses were first reported by Kino et al in 1987,1 using mice as experimental models. Subsequent reports have con­firmed the powerful immunosuppressive properties of tacrolimus in rodents, dogs and primates. These in­clude models of organ allograft rejection, ranging from skin grafts to multi-visceral transplants (see recent reviews in refs. 13-17). Tacrolimus is also effective in preventing and reversing graft-versus-host disease after experimental bone marrow transplantation. In each instance, tacrolimus has been shown to be about 10-fold more potent than CsA.

TOXICITY OF TACROLIMUS IN EXPERIMENTAL ANIMALS

The toxic effects of tacrolimus in experimental animals (rodents, rabbits, dogs and primates) have been well documented. 18 Dogs are especially suscep­tible to tacrolimus toxicity and exhibit dose-related vasculitis and intususseptions. Several groups have shown that the toxic effects of tacrolimus in nonhu­man primates are more pronounced when the drug is administered i.m. compared with orally, due pre­sumably, to the greater bioavailability achieved us­ing the former route. T acrolimus does not exhibit mutagenic activity in either in vitro or in vivo tests. Fetotoxicity has been demonstrated in rats and ter­atogenic effects have been observed in rabbits.

An important issue is whether the nephrotoxic potential of tacrolimus and CsA (or their analogs/ derivatives) correlates with the drugs' PPIase inhibi­tory activities. Recent datal? indicate that immu­nosuppressive activity and not immunophilin bind­ing or PPlase inhibitory activity determines the ability of CsA analogs to induce nephrotoxicity. It may thus be difficult to design new non nephrotoxic drugs that retain the same potent immunosuppressive activity.

INFLUENCE OF TACROLIMUS IN AUTOIMMUNE DISEASE MODELS (See Table 10.3.2 and refs. 20-43)

ExPERIMENTAL AUTOIMMUNE UVEITIS

173

Experimental autoimmune uveitis (EAU) is an organ-specific autoimmune disease of the eye that can be induced by immunization with retinal antigens, i.e., retinal soluble antigen (S-antigen) or interpho­to receptor retinoid-binding protein (IRBP). EAU is believed to be T cell mediated and a good model of autoimmune uveitis in humans. The influence of tacrolimus in EAU has been studied extensively in the Lewis rat by Mochizuki and his colleagues. Tacrolimus was found to be 10-30 times more po­tent than CsA in preventing induction of EAU when administered either from 0-5 days or from 7-12 days after S-antigen immunization. It appears that tacroli­mus is effective in suppressing on-going immuno­pathological processes, even after the disease has been initiated.20 As with CsA, the immunological unrespon­siveness induced by a 2 week course of tacrolimus (days 0-14) was found to be specific to the S-anti­gen. Whilst splenic lymphocytes from animals treated with either drug showed markedly depressed responses to S-antigen in vitro, only tacrolimus was found to significantly depress serum antibody levels. 21 EAU in­duction, as well as immune responses to S-antigen were suppressed long after cessation of tacrolimus treatmen t. 22

It has been reported that spleens of S-antigen immunized and tacrolimus-treated rats contain anti­gen-specific T suppressor (Ts) cells which, when trans­ferred to naive recipients can inhibit the induction of EAU. Moreover, Ts cells from the same donors suppress antigen-specific proliferative responses of S­antigen primed cells without influencing responses of IRBP-sensitized cells.23

Immunohistochemical studies on lymphocytes infiltrating the ocular lesions in EAU have revealed that tacrolimus reduces the absolute number of T cells, potentiates the recruitment of CDS- (Tc/s) cells, and inhibits both IL-2R expression on T cells and expres­sion of MHC class II antigens on ocular resident cells. 24

Further insight into the mode of action of tacrolimus in uveitis comes from the observation that the drug reduces intercellular adhesion molecule (lCAM-l) expression on both CD4- lymphocytes and retinal pig­ment epithelial (RPE) cells (candidate antigen pre­senting cells). Tacrolimus also inhibits binding of CD4' lymphocytes to RPE cells. 2s

In rhesus and cynomolgus monkeys, tacrolimus (0.5 mg/kg/day) administered i.m. for at least 2 weeks from 3 weeks after immunization with S-anri­gen prevented EAU. Antibody titers against S-antigen were reduced, whilst S-antigen-induced lymphocyte

174 Principles of Drug Development in Transplantation and Autoimmunity

Table 10.3.2. Experimental autoimmune diseases suppressed by tacrolimus (FK506)

Disease

Arthritis (Type II collagen-induced)

Type I diabetes

Uveoretinitis

Thyroiditis

Lupus (SLE)

Glomerulonephritis

(Nephrotoxic antiserum nephritis)

Heymann nephritis

Allergic encephalomyelitis

Autoimmune myocarditis

Experimental allergic contact dermatitis

Murine (coxsackie B3) myocarditis

Species

Rat (Lewis) Rat (Outbred) Mouse (DBA/l)

NOD mouse Cyclophosphamide

-treated NOD mouse BB rat BS rat

Rat (Lewis) Rhesus & cynomolgus

monkeys

Rat (PVG)

MRL-Ipr/lpr mouse NZB/NZW F1 mouse

Rat (Wistar)

Rat (Wi star)

Rat (Wistar) Rat (Lewis)

Rat (Lewis)

Rat (Lewis)

Farm pig

Mice (C3H/He)

a Suppresses Induction of disease b Partially effective during efferent phase of response c On day of immunization d Administered daily from 27· 120 days ot age e Effective only in induction phase f Administered from 3 weeks after immunization

Tacrolimus Dose (mg/kg/day

unless specified)

0.32a,b

2.se 2.0

2.0 mg/kgf48 hra

0.2,1,2

1.0a 25 I1g Lm.d

1.0a,e

O.Sf

2.08

2 mgh

2.5 mg/kgf48 hri

0.3 mgi

0.64

0.64a 1.0k

1.Qi

0.1,0.32,1

0.04, 0.4% topical

2.S

g Administered for 3 weeks following Inductron of disease h Administered from 8 weeks of age i From time of immunlzatron j Administered 5 days per week after immunization k Administered from day 0-13 or 56·69

Reference

Inamura et al27 (1988) Arita et a128 (1990) Takagishi et al29 (1989)

Miyagawa et aP' (1990) Carroll et aP2 (1991)

Murase et apo (1990) Nicoletti et al33 (1991)

Kawashima et al20 (1990) Fujino et al26 (1990)

Tamura et al34 (1992)

Yamamoto et al35 (1988) Takabayashi et al36 (1989)

Hara et aP7 (1990)

Okuba et aP8 (1990)

Okuba et aP8 (1990) Matsukawa et al 39 (1992)

Inamura et al 40 (1988)

Hanawa et al 41 (1992)

Meingassner & Stutz42

(1992)

Hiraoka et al 43 (1992)

The Influence of Tacrolimus on Experimental Autoimmune Disease

proliferation in vitro was unchanged or decreased dur­ing tacrolimus treatment.26

ARTHRITIS Collagen arthritis can be induced readily in many

rat strains by immunization with homologous or het­erologous native type II collagen emulsified in com­plete Freund's adjuvant (CFA). The disease is char­acterized by the development of cellular and humoral responses to type II collagen and can be transferred by sensitized spleen and lymph node cells and by antibodies to type II collagen. Inamura et al27 reported that administration of tacrolimus to Lewis rats for 12 days following immunization suppressed arthritis and inhibited both antibody and delayed-type hyper­sensitivity (DTH) skin tests to type II collagen. Failure of the animals to respond to re-immunization on day 50, but to develop experimental allergic encephalo­myelitis in response to myelin basic protein, suggested the development of long-lasting, antigen-specific un­responsiveness. 27 Studies by Arita et al2S in Sprague­Dawley rats confirmed the efficacy of tacrolimus in preventing collagen-induced arthritis; a single injec­tion of 10 mg/kg at the time of immunization sup­pressed arthritis completely and almost abolished IgG antibody and OTH responses to type II collagen. They further showed that if withheld until day 12 or 15 after immunization, the same single high dose of tacrolimus was effective in suppressing existing dis­ease and immune (humoral) responses to type II col­lagen. Moreover, pretreatment of rats with a single tacrolimus injection (10 mg/kg) (day -7 or -3) re­duced disease severity and antibody production.

Similar results concerning the prophylactic effect of tacrolimus in collagen arthritis have been obtained in mice using doses of drug 25 times lower than effec­tive doses of CsA.29 As with CsA, tacrolimus was inef­fective in treatment of established lesions in the mouse.

INSULIN-DEPENDENT DIABETES Spontaneously diabetic BB rats are considered an

excellent model for type I, insulin-dependent diabe­tes mellitus (100M). The disease shows genetic predis­position, abrupt onset of insulin-dependent ketosis­prone diabetes (60-120 days of age) associated with lymphocytic insulitis and virtually complete destruction of insulin-producing pancreatic ~ cells (Fig. 10.3.1(a, b)). Administration of tacrolimus (2 mg/ kg/day) from 30 to 120 days of age prevented the destruction of insulin-producing cells (Fig. 10.3.1 (c, d» and the development of diabetes (Fig. 10.3.2) in 20/20 BB rats during the treatment period.30 Blood glucose, renal and hepatic function tests remained nor­mal, whilst histological examination confirmed the absence of insulitis. Glucose intolerance, which has been described in BB rats given CsA was not ob­served in this study.

775

In nonobese diabetic (NOD) mice, IDDM de­velops spontaneously in about 80% of female mice between weeks 12 and 26. The disease is thought to have a CD4+ T cell dependent autoimmune patho­genesis. Administration of tacrolimus (2 mg/kg/48 hr) to female NOD mice from weeks 5-20 inhibited both the insulitis and the occurrence of diabetes (cumula­tive incidence up to 40 weeks: 86% in control mice and 23% in tacrolimus-treated animals). These ef­fects were accompanied by significant reductions in splenic CD4+ and CD8+ T cells compared with un­treated controls, suggesting that the suppression of disease activity may be linked to inhibition of cell­mediated autoimmune reactivity.3l

In both the BB rat and NOD mouse studies, the preventive effect of tacrolimus in diabetes often outlasted the duration of treatment by many weeks or in some animals permanently.

SPONTANEOUS AUTOIMMUNE Lupus DISEASE The New Zealand black/white (NZB/W) hybrid

mouse spontaneously develops nonorgan-specific, autoimmune immune complex type disease that re­sembles systemic lupus erythematosus in man. Neph­ritis and proteinuria develop within 2-3 months of age, leading to chronic renal failure and 50% mor­tality by 8-9 months. Takabayashi et aP6 reported that tacrolimus (2.5 mg/kg, 3 times per week from 12 weeks of age) prolonged the lifespan of female NZB/W Fl mice and significantly reduced proteinuria. There were, however, no differences in anti-dsDNA antibody levels or IgG subclass distribution between drug-treated animals and controls.

The MRLI Ipr mouse spontaneously develops glomerulonephritis, marked lymphoid hyperplasia, arteritis and chronic polyarthritis, with 50% morral­ity at about 5 months. Takabayashi et aP6 reported similar effects of tacrolimus on survival, proteinuria and anti-dsDNA antibody levels in MRLI Ipr female mice (given 2.5 mg/kg tacrolimus 3 times per week from 8 weeks old) to those observed in NZB/W F 1

hybrids. Compared with untreated MRLI Ipr controls, minimal glomerulonephritis with only mild prolifera­tion of endothelial and mesangial cells was noted, whilst immunoglobulin and C3 deposits were restricted mainly to the mesangia. Using a similar tacrolimus treatment protocol, Yamamoto et aP5 found that in­hibition of disease activity in MRLI Ipr mice was ac­companied by significant reductions in serum anti­ssONA and anti-dsONA activities.

ExPERIMENTAL AUTOIMMUNE GLOMERULONEPHRITIS

There are several ways of inducing immune-me­diated nephritis in experimental animals. Heymann's nephritis is a membranous type of chronic glome­rulonephritis with glomerular subepithelial immune

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The Influence of Tacrolimus on Experimental Autoimmune Disease 177

FK 506 2 mg/kg (n=20)

100O===========i===========~r---------~1

Fig. 10.3.2. Inhibition of development of diabetes in tacrolimus (FKS06)­treated BB rats.

tn ca E c ca .2 -Q) .Q

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Days of age

deposits induced by autologous antibody against re­nal tubular brush border (TBB) antigens. It is in­duced in rats by footpad immunization with TBB antigen in FCA. Another type of glomerulonephritis (nephrotoxic antiserum nephritis or Masugi nephritis) is induced by the intravenous injection of (rabbit) antibody to rat glomerular basement membrane (GBM). Okuba et aP8 have shown that tacrolimus (0.64 mg/kg) given for 14 days from the time of TBB immunization completely suppresses development of Heymann's nephritis. They have also found that tacrolimus prevents the autologous phase (i.e., pro­duction of antibody against rabbit gamma globulin fixed on the GBM) of nephrotoxic antiserum nephritis. In both models, the rats remained tolerant to the immunizing antigens but not to other antigens for at least 14 weeks after drug withdrawal. The induc­tion by tacrolimus of long lasting, antigen-specific unresponsiveness is similar to the effects of the drug in EAU and collagen-induced arthritis (see above). The underlying mechanism(s) has not been elucidated, but possible explanations include clonal deletion or the induction of anti-idiotypic antibody or antigen­specific suppressor cells.

Glomerulonephritis can also be induced by preimmunization of rats with normal rabbit IgG and the subsequent i.v. injection of rabbit anti-GBM antibody (accelerated nephrotoxic serum glomerulo­nephritis). I n this model, tacrolimus suppresses the autologous response co rabbit IgG and reduces sub­stantially the glomerular injuryY

ExPERIMENTAL AUTOIMMUNE THYROIDITIS Experimental autoimmune thyroiditis can be in­

duced in female PVG/c rats by early thymectomy followed by a course of sublethal total body irradia­tion (TBI). This results in progressive, lymphocytic infiltration of the thyroid, follicular obliteration and production of anti-thyroglobulin autoantibodies. Ad­ministration of tacrolimus (2 mg/kg/day) from week 8 after the last dose of TBI significantly decreases the disease severity and is associated with reversal of the splenic CD4+: CDS+ T cell ratio. 34

ExPERIMENTAL ALLERGIC ENCEPHALOMYELITIS Experimental allergic encephalomyelitis (EAE) is

induced in susceptible rat strains by a single injec­tion of myelin basic protein (MBP) in FCA. An acute paralytic disease develops within 10-14 days after immunization with features similar to human acute inflammatory central nervous system disease. Tacroli­mus (1 mg/kg 5 days per week for 2 weeks from the time of immunization) prevented development of EAE for at least 50 days and completely suppressed both antibody production and cell-mediated immunity to MBP. The effectiveness of the drug, however, was considerably reduced when treatment was delayed until 7 days after immunization;IO It has been observed that the adoptive transfer of EAE can be suppressed by in vitro treatment of spleen cells with 1 nM tacrolimus, IOO-fold lower than (he effective concentration of CSA.44

178 Principles of Drug Development in Transplantation and Autoimmunity

COMBINED T AND B CELL DIRECTED THERAPY OF SPONTANEOUS AUTOIMMUNE DISEASE USING TACROLIMUS AND LOW DOSE CYCLOPHOSPHAMIDE

Autoimmune diseases in which autoantibodies and immune complexes appear to playa dominant role, such as SLE, various vasculitides and glomerulo­nephritis have not responded as favorably to CsA as those disorders that are thought to be predominantly T cell driven. The current management of the former disorders includes treatment with varying doses of corticosteroids and for certain organ system involve­ment, use of cyclophosphamide (Cy):15 Responses are frequently only partial however, and often associated with severe side effects, including life-threatening infections.

The search for improved strategies of immuno­suppressive therapy includes the evaluation of com­binations of drugs with complementary actions. In principle, two or more agents can be used together at reduced dosage to achieve additive or synergistic therapeutic effects while minimizing toxicity. Recently, it has been shown that combination of either of the T cell suppressants, CsA46 or tacrolimus47 with the alkylating agent CY can effectively control the strong antibody and cell-mediated responses evoked by con­cordant xenografts in experimental animals. Moreover, this approach has been found to prolong graft sur­vival much longer than drug mono therapy. There­fore, it is possible that the combination of CsA or tacrolimus with CY may prove valuable in the treat­ment of autoimmune disorders in which both T cells and antibody-producing B cells are thought to play important pathogenic roles.

Mice homozygous for the lpr gene that encodes a defective allele of the apoptosis regulatory gene Fas48

spontaneously develop an aggressive T cell dependent

autoimmune disease that resembles human SLE.49-51 MRL-lpr//pr mice also develop high levels of autoan­tibodies, associated with glomerulonephritis and vasculitis, interstitial pneumonitis, arthritis (in some, but not all colonies) and premature death. It has been shown (see above) that high doses of tacrolimus can inhibit disease progression and prolong survival. The incidence of proteinuria and anti-DNA autoantibodies may however, remain high. 35•36 Since a combination of tacrolimus and CY has been shown to be effective in controlling both T cell and antibody-mediated immune responses in vivo, we have examined the effect of this drug combination on the development of au­toimmune disease in MRL-lpr mice.

Groups of female MRL-/pr mice received either saline or tacrolimus (2 mg/kg i.p) thrice weekly, CY (20 mg/kg) once monthly, or both drugs from 8 weeks of age. Median survival for untreated and CY-treated mice was 26 weeks and for tacrolimus and tacrolimus + CY-treated groups was> 44 weeks. 52 Severity of skin lesions and lymph node hyperplasia was mark­edly reduced by the drug combination, whereas ei­ther drug alone was less effective (Table 10.3.3). Tacrolimus or CY alone delayed the onset of pro­teinuria, but by 24 weeks all of these animals were positive. In contrast, drug combination reduced the prevalence and severity of proteinuria throughout the 44 weeks of study (Fig. 10.3.3). Sequential monitor­ing of peripheral blood lymphocytes revealed that combination therapy but not mono therapy markedly reduced the proportion of atypical CD3+ B220+ and CD3+CD4-CD8- T cells. Serum levels of anti-dsDNA antibodies were reduced in all treatment groups. Analysis of tissue from saline-treated mice that died spontaneously showed as anticipated, evidence of ex­tensive diffuse interstitial disease, diffuse proliferative glomerulonephritis with crescents, and vasculitis (Fig. 10.3.4(a)). Similarly, the CY-treated mice showed

Table 10.3.3. Influence of tacrolimus (FK506) and tacrolimus + cyclophosphamide (CY) on the clinical disease index (lymphoid hyperplasia, exudative dermatitis and ear necrosis) in female MRL-Ipr mice

Treatment Saline Tacrolimus CY Tacrolimus + CY Age (weeks)

8 0(10) 0(10) 0(6) 0(10) 12 0(10) 0(10) 0(6) 0(10) 16 1.4 ± 0.9(9) 1.2 ± 0.4(10) 1.2 ± 0.4(4) 1.0 ± o.sa(lO) 20 1.8 ± 0.6(7) 0.9 ± 0.4(10) 1.8 ± 0.9(4) 0.6 ± 0.6b( 10) 24 2.5 ±0.6(S) 1.9± 0.8(10) 1.8 ± 0.9(4) 0.9 ± 0.9b(lO) 33 3.0(1) 2.8 ± 1.2(9) 3.5(1) 1.3 ± 1.2( 10) 44 No survivors 2.6 ± 1.3(7) 2.5(1 ) 1.3 ± 1.3(7)

0-, no physical signs; clinical involvement scored 14 (most severe). Total score tor each group was divided by the total number oi mice alive (in parentheses) when determinations were made. d, p < 0.05; b, p < 0.01 compared with saline-treated control. Reproduced with permission irom Woo J et al. elin Exp Immunol 1995; 100: 118.125.52

The Influence of Tacrofimus on Experimental Autoimmune Disease

Proteinuria in MRL-lpr/lpr Mice

Albumin Level ml:/dL

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179

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The Influence of Tacrolimus on Experimental Autoimmune Disease

severe interstitial and glomerular disease with vasculitis (Fig. lO.3.4(b». In contrast. the kidneys of mice treated with tacrolimus or tacrolimus + Cy (Figs. 10.3.4 (c.d)). sacrificed at 44 weeks showed only moderate focal interstitial and glomerular changes. There was no significant difference in the severity of renal dis­ease between the tacrolimus and tacrolimus + Cy groups. These data demonstrate the improved effi­cacy of combined T and B cell directed immuno­suppression in murine lupus. associated with marked inhibition of atypical T cells within the affected lym­phoid tissue. Since Cy is used currently in the treat­ment of SLE, the combined use of these powerful immunosuppressants for the more effective control of this debilitating disease is worthy of further evalu­ation.

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